Appearance and taste are the main criteria used by consumers to assess a beer, but also other beverages such as e.g. wine, juice or mixed beverages. The so-called “flavor”, which results from the synergy between the gustatory and olfactory sensations, should therefore remain as constant as possible for the entire duration up until the specified best before date. However, the natural aging process of a beverage, preferably a beer, works against this. For this reason, the flavor stability is becoming increasingly important as an essential quality feature of beer.
The oxidative flavor stability is substantially dependent on the extent to which a beer is able to prevent the formation of primary oxygen radicals (O−2.; OH.; H.; HO2.) and is accordingly influenced by the endogenous antioxidative potential (EAP) of a beer, which is based on reducing compounds such as e.g. sulfur dioxide (SO2), Maillard reaction products, ascorbic acid and phenolic ingredients.
The radicals produced in the course of beer aging are usually initiated via various forms of activated oxygen, which therefore plays an important role in the onset of the aging flavor. Among the primary oxygen radicals, particular mention should be made of the hydroxyl radical produced via the Fenton and Haber-Weiss reaction; this radial assumes a key position in beer aging. If the production of hydroxyl radicals can be reduced or even prevented, it is possible to delay the onset of the aging flavor, which is brought about in particular by carbonyl compounds, and especially aliphatic aldehydes [5, 6, 14, 15].
Beers are able to prevent the formation of hydroxyl radicals for a certain period of time before the generation of radicals proceeds unhindered. Under identical external conditions (e.g. temperature, oxygen contact, etc.) and with constant conditions in a beer sample (e.g. pH, dissolved oxygen, etc.), the period of time over which the generation of radicals can be prevented or delayed is directly related to the existing EAP.
With the aim of being able to make a statement about the flavor stability that can be expected of a beer, for some years use has been made of electron spin resonance (ESR) spectroscopy. In this process, the so-called “lag time” of a beer is determined via an experimentally accelerated (“forced”) beer aging at increased temperatures (“forcing test”, usually at 60° C.). The lag time value corresponds to the point in time from which the generation of radicals in the beer proceeds unhindered under defined experimental conditions, and the value of the lag time determined using this measurement method is considered to be a criterion for the EAP of the beer [1-11].
In this measurement method, which was first described by Kaneda et al. [12] and was further developed by Uchida et al. [1], the property of ESR spectroscopy is used to selectively detect radicals with a high level of sensitivity in the complex system comprising beer with a large number of different ingredients. Since radicals in aqueous solutions usually have only a very short life span, it is necessary to use a radical scavenger (“spin trap”) which is able to accumulate diffusible radicals. With the spin trap reagent N-tert-butyl-α-phenylnitrone (PBN) selected by Uchida et al. [1], a much more stable nitroxide radical is produced which accumulates to a sufficient extent over time and can thus be detected by ESR spectroscopy on the basis of its spectral characteristic [13].
The mechanism of lag time measurement according to the known method has been interpreted so that it is the hydroxyl radicals which are important with regard to oxidative beer aging, and the time-dependent formation of said radicals is detected using the spin trap reagent PBN during the forcing test.
Besides ESR spectrometers in research equipment, the company Bruker (Karlsruhe) offers a table-top device called “e-scan” (small X-band ESR spectrometer), by means of which the lag time can be determined using the method described above in the laboratories of the beverage-producing industry.
However, the described method exhibits significant weaknesses and falsifications. In this connection, reference is made in particular to a pH effect which is brought about by the spin trap reagent PBN that is used. This effect leads to the pH of a beer sample rising during the lag time measurement as a function of the PBN concentration used. According to Bishop et al. [19] and Millero et al. [20], a rise in the pH, in particular in the pH range >4.5 (Fenton reaction), leads to excessive acceleration of the generation of hydroxyl radicals.
The rise in pH dependent on the PBN concentration (pH effect) during the lag time measurement accelerates the generation of radicals in the beer, and the existing EAP is used up increasingly quickly over the duration of the lag time measurement. The point in time at which unhindered radical generation starts (measured lag time) deviates to a correspondingly large extent from the conditions that actually exist in the beer. Even for this reason alone, the lag time measurement according to the previous method is not readily suitable for correctly determining the EAP of a beer.
To complicate matters, added to this is the fact that the degree of falsification caused by the addition of PBN with regard to the time of unhindered radical generation (pH effect) is additionally influenced by the existing EAP of a beer. This can be explained by the fact that the pH effect on the generation of radicals within the lag time is different for beers with different EAPs. As a result, the generation of radicals within a high lag time is influenced for a greater length of time and to a greater extent—since higher pH values are achieved—towards an increased generation of hydroxyl radicals, and the difference between the measured lag time and the conditions that actually exist in the beer increases accordingly.
Another falsification factor is the fact that many ingredients with an antioxidative effect (e.g. phenolic substances, etc.) are pH-dependent with regard to their action. Since there are many falsifications and the differences in the case of beers with a medium to high oxidative beer stability are up to 600%, for many or most beers it is no longer possible to establish any connection to the EAP. The lag time measurement according to the previous procedure is unsuitable for determining the EAP of the beers, and the results obtained have recently led to corresponding misinterpretations in various publications. Not only are the effects of the beer ingredients on the oxidative beer stability greatly falsified, but also the brewing measures based on these measurements to improve the oxidative beer stability are falsely estimated on the basis of the results.
In the prior art [16, 17], there are also indications that the radicals detected with PBN are in the main not hydroxyl radicals themselves but rather that the hydroxyethyl radicals produced as the result of a subsequent reaction of the hydroxyl radical with the ethanol of the beer are involved to a greater extent than the previously assumed few percent in the measured signal intensity. The reason given for this was a higher stability of the stabilized hydroxyethyl PBN spin adducts [17]. The question as to which radicals ultimately play a role in the lag time determination is particularly important not just with regard to the method itself, but may also be particularly useful for the further clarification of the aging processes, which are not yet fully known.
There is therefore a need for an improved method for assessing the flavor stability of beer and other oxidative beverages.
It was therefore an object of the present invention to provide a method which can be used to determine the EAP of beer and other beverages in a more reliable manner than before.